Assay method and kit therefor

ABSTRACT

The invention relates to a method of determining an analyte in a sample, especially a high concentration analyte, comprises the steps of:  
     a) contacting the sample with a specified amount of a receptor which binds specifically to the analyte to form an analyte/receptor complex, said specified amount of receptor being in excess of that required to bind all analyte in the sample,  
     b) isolating on a solid phase a specified fraction of the amount of receptor contacted with the analyte, including analyte/receptor complex and unreacted receptor,  
     c) detecting the amount of analyte/receptor complex in said isolated specified fraction, and  
     d) from the detected amount analyte/receptor complex, determining the concentration of analyte in the sample.  
     The invention also relates to test kits for carrying out the method.

FIELD OF THE INVENTION

[0001] The present invention relates to a method of quantitatively orsemi-quantitatively determining an analyte in a sample, especially ahigh concentration analyte.

BACKGROUND OF THE INVENTION

[0002] For qualitative and quantitative determination of an analyte in asample, a so-called sandwich assay is often used, wherein two receptorsdirected against different epitopes of the analyte are incubated with asample containing the analyte, one of the receptors being detectable,e.g. through a label conjugated thereto. In a heterogeneous assayformat, the second receptor is immobilized (e.g. coupled) to a solidphase or provided with a binder component, such as biotin, capable ofbinding to the solid phase, such as an avidin- or streptavidin-coatedsolid phase.

[0003] Especially in case the analyte is present in the sample in a highconcentration, it is customary to dilute the sample before performingthe assay to avoid the use of large and often costly amounts ofimmobilized receptor and labelled receptor, respectively, or to avoidtechnical difficulties where large amounts of receptors cannot be used.Such dilution is not only laborious but also introduces an additionalsource of error into the assay.

[0004] There is therefore a need of an assay procedure that avoids thenecessity of dilution.

SUMMARY OF THE INVENTION

[0005] It is an object of the present invention to provide a method ofperforming a heterogeneous sandwich assay which permits thedetermination of even a high concentration analyte in a sample withoutthe need to dilute the sample.

[0006] It is another object of the invention to provide a method ofperforming a heterogeneous sandwich assay which reduces the amounts ofcapturing and detection reagents used.

[0007] It is still another object of the invention to provide test kitsfor carrying out the method.

[0008] In one aspect of the present invention there is thereforeprovided a method of determining an analyte in a sample, especially ahigh concentration analyte found in concentrations >1 nmole/liter,comprising the steps of:

[0009] a) contacting the sample with a specified amount of a receptorwhich binds specifically to the analyte to form an analyte/receptorcomplex, said specified amount of receptor being in excess of thatrequired to bind all analyte in the sample,

[0010] b) isolating on a solid phase, preferably a matrix such as amembrane strip, a specified fraction of the amount of receptor contactedwith the analyte, including analyte/receptor complex and unreactedreceptor,

[0011] c) detecting the amount of analyte/receptor complex in saidisolated specified fraction, and

[0012] d) from the detected amount analyte/receptor complex, determiningthe concentration of analyte in the sample.

[0013] In another aspect of the present invention there is provided atest kit for determining an analyte in a sample, comprising (i) aspecified amount of a receptor substance having a first part which bindsspecifically to the analyte, and (ii) a solid phase member havingimmobilized thereon a ligand which binds specifically to a second partof the receptor, the amount of said ligand on the solid phase memberbeing less than said specified amount of the receptor substance.

[0014] In still another aspect of the present invention there isprovided a test kit for determining an analyte in a sample, comprising(i) a specified amount of a receptor substance having a first part whichbinds specifically to the analyte, only a specified fraction of theamount of receptor substance having a second part capable of binding toa specific ligand, and (ii) a solid phase member having said specificligand immobilized thereon.

[0015] In yet another aspect of the present invention there is provideda test kit for determining an analyte in a sample, comprising (i) afirst specified amount of a receptor substance, and (ii) a solid phasemember having immobilized thereon a second specified amount of thereceptor substance.

[0016] While it is preferred to use the method and test kit forquantitative determination of analytes of interest, they may also beused for semi-quantitative and qualitative determinations.

DETAILED DESCRIPTION OF THE INVENTION

[0017] The essence of the present invention resides in binding allanalyte present in a sample to an analyte-specific receptor, isolating aminor fraction of the analyte-receptor complex formed on a solid phase,detecting the amount of isolated analyte-receptor complex, and from thisdetected amount of analyte on the solid phase determining the totalamount of analyte in the sample. According to the invention, this may beaccomplished in various ways.

[0018] In one embodiment of method of the invention, all analyte isbound by contacting the analyte-containing sample with a solutioncontaining an excess of a first receptor (R1) which in addition toaffinity to the analyte has affinity to a ligand (L), whereupon a minorfraction of the analyte-receptor complex is bound to a solid phasehaving the ligand (L) immobilized thereto. This binding of the minorfraction may be achieved by either (i) using a limited (specified)amount of ligand (L) to extract a fraction of the analyte-receptorcomplex (and unreacted receptor), or (ii) by using a first receptor (R1)only a minor (specified) fraction of which is capable of binding to theligand (L) to extract the desired fraction of the analyte-receptorcomplex (and unreacted receptor). In the latter case (ii), the amount ofimmobilized ligand (L) is usually in excess of the amount of the firstreceptor capable of binding to the ligand (L). The amount ofanalyte/receptor complex bound to the solid phase is then detected,usually by contacting the solid phase with a detecting agent in the formof a labelled binder for the analyte, such as a labelled second receptor(R2).

[0019] In the first case (i) above, the amount of immobilized ligand (L)that can bind to the analyte-specific receptor (R1) is a specifiedfraction of the amount of analyte-specific receptor (R1) contacted withthe sample, and therefore the ratio of detected analyte on the solidphase to the total amount of analyte in the sample will correspond tothe ratio of immobilized analyte-binding ligand (L) to the total amountof added receptor (R1), thereby permitting the analyte concentration inthe sample to be calculated.

[0020] In the second case (ii) above, the amount of analyte-specificreceptor (R1) that can bind to immobilized ligand (L) is a specifiedfraction of the total amount of receptor (R1), and therefore the ratioof detected analyte on the solid phase to the total amount of analyte inthe sample will correspond to the ratio of analyte-specific receptor(R1) capable of reacting with ligand (L) to the total amount of receptor(R1), thereby permitting the analyte concentration in the sample to becalculated.

[0021] The term “receptor” as used herein refers to activeanalyte-binding receptor, and, where relevant, active ligand-bindingreceptor, respectively, and is not meant to include such receptor in aninactive or non-binding state. Likewise, the term receptor-bindingligand refers to active receptor-binding ligand and is not meant toinclude such ligand in an inactive or non-binding state.

[0022] The term “amount” as used herein usually means binding capacity.Thus, for example, when it is stated that the amount of analyte-specificreceptor is in excess of the amount of analyte, it means that there ismore analyte-specific receptor than necessary to bind all analyte.Usually, there is a 1:1 reaction ration between e.g. the analyte and theanalyte-specific receptor, or between the analyte-specific receptor andthe immobilized receptor-binding ligand. In such a case, the bindingcapacities of the respective species correspond to their molar amounts.Other reaction ratios are, however, also possible. For example, theimmobilized ligand may be capable of binding more than oneanalyte-specific receptor.

[0023] In another embodiment of method of the invention, the sample iscontacted with analyte-specific receptor (R1) provided both in solutionand, in a minor fraction, immobilized to a solid phase, therebypermitting a minor fraction of analyte present in the sample to be boundto the solid phase. If the ratio of the amount of receptor (R1) insolution to the amount of immobilized receptor (R1) is known, theanalyte concentration in the sample may be calculated from the detectedamount of analyte bound to the solid phase.

[0024] It is readily seen that the above procedure gives the same effectas diluting the sample. In addition to the dilution step being avoided,which, of course, is of advantage to the operator, one obtains aconsiderable saving in reagents, i.e. both the reagent for capturing theanalyte on the solid phase and the detecting agent, the latter oftenbeing costly. In this connection, it is also to be noted that in theassay of the invention, the reaction between analyte and receptor takesplace in solution where almost all receptors are active rather than at asolid phase surface as in a corresponding conventional assay where onlyabout 10-20% of immobilized receptor will react (Butler, J. E., et al,Molecular Immunology, Vol. 30, No. 13, pp. 1165-1175, 1993).

[0025] The required ratio between the total binding capacity ofanalyte-specific receptor contacted with the sample and (i) the bindingcapacity of receptor-binding ligand that is immobilized to the solidphase when this is limited, or (ii) the ligand-binding capacity of theanalyte-specific receptor when this is limited, is readily determined bythe skilled person depending inter alia on the particular analyte to bedetermined and the particular assay format used and may be chosen withinwide limits. Usually, this ratio is from about 2:1 to about 1000:1,especially from about 5:1 to about 100:1, preferably more than about10:1, more preferably more than about 20:1.

[0026] The excess of analyte receptor relative to the amount of analytein the sample is also readily determined by the skilled person for eachspecific case.

[0027] The receptor contacted with the sample is usually of the dualreceptor or bireactive binder type having one part that specificallybinds to the analyte and another part which specifically binds to theligand immobilized on the solid phase surface. The analyte binding partmay, for example, be an antibody (monoclonal or polyclonal) or an activefragment thereof (including recombinant antibodies and fragments) ornucleic acids whereas the ligand-binding part may be one member of aspecific binding pair. Exemplary such specific binding pairs includeimmunological binding pairs, such as antigen-antibody andhapten-antibody, biotin-avidin or -streptavidin, lectin-sugar,hormone-hormone receptor, and nucleic acid duplex. For example, thesolid phase may have streptavidin immobilized thereto, and the receptorfor the analyte may be biotinylated. To avoid immunoprecipitation athigh analyte concentrations, it may be preferable to use monovalentreceptors.

[0028] While the analyte preferably is a molecule present inconcentrations >1 nmole/liter in a sample, the analyte may, of course,be any substance for which there exists a naturally occurringanalyte-specific binding member or for which an analyte-specific bindingmember can be prepared.

[0029] Analyte that has been captured by the solid phase is usuallydetected by reaction with a labelled specific binder for the analyte.Such a labelled binder may be a conjugate comprising a detectable labelcovalently or non-covalently attached to the specific binding member,“label” referring to any substance which is capable of producing asignal that is detectable by visual or instrumental means, particularlya fluorophore or chromophore.

[0030] The sample is usually of biological origin, for example blood(serum, plasma, whole blood), saliva, tear fluid, urine, cerebrospinalfluid, sweat, etc. The invention is, of course, also applicable to othertypes of samples, such as fermentation solutions, reaction mixtures,etc. Especially, however, the sample is an undiluted serum or wholeblood sample.

[0031] While the present invention is generally applicable, it mayadvantageously be used in solid phase assays of the immunochromatograpictype. Such assays use a device comprising a plate-shaped flow matrix ofbibulous material, usually a membrane strip, such as of cellulosenitrate or glass fiber, in which liquid can be transported laterally(i.e. in the plane of the strip) by capillary forces in the membrane.The membrane usually has a sample application zone, and a detection (orreaction) zone downstream of the sample application zone. In thedetection zone, usually a capturing reagent for the analyte isimmobilized. To conduct an assay, the application zone is contacted withthe liquid sample to be assayed for the analyte of interest. The deviceis maintained under conditions sufficient to allow capillary action ofliquid to transport the analyte of interest, if present in the sample,through the membrane strip to the detection zone where the analyte iscaptured. The capillary liquid flow is usually insured by an absorbingpad or the like at the downstream end of the strip. A detection reagent,usually labelled, is then added upstream of the detection zone andinteracts with captured analyte in the detection zone, and the amount ofcaptured analyte is measured. Often, the detection reagent ispre-deposited in or on the membrane strip, e.g. in the form ofdiffusively movable particles containing fluorophoric or chromogenicgroups, either upstream of the sample application zone or between thesample application zone and the detection zone.

[0032] In an immunochromatographic assay according to the invention, thereceptor is added to the sample either before applying the sample to themembrane strip, or may be pre-deposited in or on the membrane stripupstream of the detection zone.

[0033] A test kit for carrying out the method of the invention maycomprise such a membrane having (i) immobilized in or on the membrane aligand which binds specifically to the receptor, and (ii) dissolvablypre-deposited in or on the membrane a specified amount ofanalyte-specific receptor. The amount of the ligand on the solid phasemember is less, and usually considerably less than that required to bindthe specified amount of the receptor substance.

[0034] In another embodiment of test kit, only a fraction of theanalyte-specific receptor is capable of binding to the immobilizedligand. Such a kit may comprise (i) immobilized in or on a membrane aligand which binds specifically to the receptor, and (ii) dissolvablypre-deposited in or on the membrane a specified amount ofanalyte-specific receptor substance, only a specified fraction of whichis capable of binding to the immobilized ligand.

[0035] Still another embodiment of test kit may comprise (i) dissolvablypre-deposited in or on a membrane a first specified amount ofanalyte-specific receptor substance, and (ii) immobilized in or on themembrane a second specified amount of the analyte-specific receptorsubstance.

[0036] In an alternative embodiment, the solid phase is a solid phasewell, such as a microtiter plate well. Such of test kit may comprise asolid support having one or more wells with the second amount of analytebinding receptor immobilized therein and with the first amount ofanalyte-binding receptor dissolvably pre-deposited in the well or inclose contact with the well.

[0037] In the following, the invention will be illustrated in moredetail by a specific non-limiting Example.

EXAMPLE 1 Immunoassay for C-reactive Protein (CRP) in Undiluted SerumSamples Measuring Range 10-200 mg/l

[0038] Principle

[0039] Sample is mixed with biotinylated anti-CRP-fab in excess and themixture is applied to a test strip having a deficient amount ofstreptavidin in the reaction zone. After an intermediate wash, anti-CRPfluorophore-conjugate is added and after a wash, conjugate that hasbound to the reaction zone is measured. Since only a small part of thebiotinylated anti-CRP-fab can bind to the reaction zone the consumptionof the fluorophore conjugate is reduced considerably.

[0040] Test Strips

[0041] 5×48 mm nitrocellulose membranes (Whatman, porosity 8 μm) on apolyester backing were used. The strips had a sample application zone atone end and a downstream reaction zone with immobilized streptavidin inan amount capable of binding approximately 6% of biotinylated anti-CRPadded in the assay procedure.

[0042] Samples

[0043] CRP-containing samples of varying CRP concentration were preparedfrom a 200 mg/l of recombinant CRP (Fitzgerald) in hCRP depleted serum.

[0044] Procedure

[0045] 15 μl of biotinylated anti-CRP-fab (monovalent fab-fragment ofmonoclonal antibody) and 15 μl of CRP-containing serum were mixed andthe mixture was applied to the application zone of the membrane strip.The amount of biotinylated anti-CRP-fab was 3 μg per test strip, whichis a 2× molar excess of anti-CRP in relation to the standard 200 mg/lCRP. After an intermediate wash with 15 μl of test buffer (50 mM boratebuffer pH 8.0, 3% BSA, 5% sucrose, 0.15 M NaCl, 0.005% CaCl₂, 0.05%NaN₃), 15 μl of detection conjugate solution [3 μg of anti-CRPmonoclonal antibody (Fitzgerald) coupled to 0.1 μmTransFluoSpheres-SO₄/CHO (633/720 nm) (Molecular Probes Inc.), the abovetest buffer] were added, followed by wash with 2×15 μl of test buffer.The fluorescence of the strip was then measured. The results are shownin Table 1 below. TABLE 1 CRP conc. Peak area obtained (mg/l) (V × mm) 0 0.08  0 0.07 10 2.56 10 2.50 30 3.62 30 4.01 100  5.24 100  4.87 200 6.28 200  5.82

EXAMPLE 2 (comparative) Immunoassay for CRP in serum samples diluted1/20 Measurement range 10-200 mg/ml

[0046] Principle

[0047] Sample is diluted in test buffer and applied to test stripshaving an excess of anti-CRP in the reaction zone. Anti-CRPfluorophore-conjugate is then added followed by a wash, whereuponconjugate that has bound to the reaction zone is measured. Sampledilution is necessary to avoid unreasonably large amounts of anti-CRP inthe reaction zone as well as in the detection conjugate.

[0048] Test strips

[0049] 5×48 mm nitrocellulose membranes (Whatman, porosity 8 μm) on apolyester backing were used. The strips had a sample application zone atone end and a downstream reaction zone with 2.6 μg immobilized anti-CRPmonoclonal antibody (Fitzgerald), which is a 13x molar excess inrelation to a standard 10 mg/ml CRP serum.

[0050] Samples

[0051] containing samples of varying CRP concentration were preparedfrom a recombinant CRP (Fitzgerald) in hCRP depleted serum.

[0052] Procedure

[0053] 15 82 l of CRP-containing serum diluted 1/20 in test buffer (50MM borate buffer pH 8.0, 3% BSA, 5% sucrose, 0.15 M NaCl, 0.005% CaCl₂,0.05% NaN₃) were applied to the application zone of the membrane strip.Then, 15 μl of detection conjugate solution [anti-CRP monoclonalantibody (Fitzgerald) coupled to 0.1 μm TransFluoSpheres-SO₄/CHO(633/720 nm) (Molecular Probes Inc.), the above test buffer] were added,the amount of anti-CRP conjugate being 3 μg per test strip which was a15× molar excess in relation to the highest standard value. Theconjugate addition was followed by a wash with 15 μl of test buffer. Thefluorescence of the strip was then measured. The results are shown inTable 2 below. TABLE 2 CRP conc. Peak area obtained (mg/l) (V × mm)  00.41  0 0.60 10 7.51 10  7.130 20 8.86 20 9.42 40 11.97  40 10.67  8011.70  80 12.91  200  14.27  200  14.16 

[0054] The above Examples 1 and 2 thus demonstrate that it is possibleto run an assay on undiluted high concentration samples without usinghuge amounts of reagents when using the methodology of the presentinvention.

1. A method of determining an analyte in a sample comprising the stepsof: a) contacting the sample with a specified amount of a receptor whichbinds specifically to the analyte to form an analyte/receptor complex,said specified amount of receptor being in excess of that required tobind all analyte in the sample, b) isolating on a solid phase aspecified fraction of the amount of receptor contacted with the analyte,including analyte/receptor complex and unreacted receptor, c) detectingthe amount of analyte/receptor complex in said isolated specifiedfraction, and d) from the detected amount analyte/receptor complex,determining the concentration of analyte in the sample.
 2. The methodaccording to claim 1 in which the sample has a high concentration. 3.The method according to claim 1 or claim 2 in which the sample isundiluted.
 4. The method according to claims 1 to 3, wherein isolatingsaid specified fraction of the amount of receptor contacted with thesample on the solid phase comprises providing a solid phase havingbinding sites for the receptor, and after contacting the sample, or analiquot thereof, with a liquid phase containing the receptor, bindingsaid specified fraction of receptor to the solid phase.
 5. The methodaccording to claim 4, wherein the whole amount of receptor hasreactivity towards said binding sites on the solid phase, and thereceptor-binding capacity of the solid phase is less than thesolid-phase-binding capacity of receptor contacted with the sample. 6.The method according to claim 4, wherein only a specified fraction ofthe amount of receptor contacted with the sample has reactivity towardssaid binding sites on the solid phase.
 7. The method according to claims1 to 3, wherein isolating said specified fraction of the amount ofreceptor on the solid phase comprises contacting the sample with aspecified amount of receptor, a specified fraction of which amount isimmobilized to said solid phase and the remaining amount of receptorbeing in a liquid phase.
 8. The method according to any one of claims 1to 6, wherein the receptor comprises a first part that bindsspecifically to the analyte, and a second part that binds to the solidphase.
 9. The method according to claim 8, wherein the solid phasebinding part of the receptor comprises one member of a specific bindingpair, and the other member of the binding pair is immobilized to thesolid phase.
 10. The method according to any one the preceding ofclaims, wherein in step c) the analyte/receptor complex is detected by alabelled detection reagent which binds specifically to the analyte. 11.The method according to any one of the preceding claims, wherein theratio between said isolated fraction of the amount of activeanalyte-binding receptor and the total amount of active analyte-bindingreceptor contacted with the sample is in the range of from about 1:2 toabout 1:1000, preferably from about 1:5 to about 1:100, particularly nomore than about 1:20.
 12. The method according to any one of thepreceding claims, wherein said solid phase binding sites for thereceptor are immobilized in a reaction zone of a flow matrix, preferablya lateral flow matrix, such as a membrane strip.
 13. The methodaccording to any one of the preceding claims, wherein the receptor is anantibody or an immunoactive fragment thereof.
 14. The method accordingto any one of the preceding claims, wherein the detection reagent is anantibody or an immunoactive fragment thereof.
 15. The method accordingto any one of the preceding claims, wherein the detection reagent islabelled by a fluorophore or a chromophore.
 16. The method according toany one of the preceding claims, wherein the specific binding pair isbiotin-avidin or biotin-streptavidin.
 17. The method according to anyone of the preceding claims, wherein the sample is an undiluted serumsample.
 18. The method according to any one of claims 1 to 16, whereinthe sample is an undiluted whole blood sample.
 19. A test kit fordetermining an analyte in a sample, comprising a specified amount of areceptor substance having a first part which binds specifically to theanalyte, and a solid phase member having immobilized thereon a ligandwhich binds specifically to a second part of the receptor, thereceptor-binding capacity of said ligand on the solid phase member beingless than the ligand-binding capacity of said specified amount ofreceptor substance.
 20. The test kit according to claim 19, wherein theratio between the receptor-binding capacity of ligand immobilized on thesolid phase and the ligand-binding capacity of the analyte-specificreceptor substance is in the range of from about 1:2 to about 1:1000,preferably from about 1:5 to about 1:100, particularly no more thanabout 1:20.
 21. The test kit according to claim 19 or 20, comprising alateral flow membrane strip having said receptor-binding ligandimmobilized in or on a reaction zone of the membrane and having saidanalyte-binding receptor substance dissolvably pre-deposited in or onthe membrane upstream of the reaction zone.
 22. A test kit fordetermining an analyte in a sample, comprising a specified amount of areceptor substance having a first part which binds specifically to theanalyte, only a specified fraction of the amount of receptor substancehaving a second part capable of binding to a specific ligand, and asolid phase member having said specific ligand immobilized thereon. 23.The test kit according to claim 22, wherein the ratio between the amountof ligand-binding analyte-specific receptor and the total amount ofanalyte-specific receptor is in the range of from about 1:2 to about1:1000, preferably from about 1:5 to about 1:100, particularly no morethan about 1:20.
 24. The test kit according to claim 22 or 23,comprising a lateral flow membrane strip having said receptor-bindingligand immobilized in or on a reaction zone of the membrane and havingsaid analyte-binding receptor substance dissolvably pre-deposited in oron the membrane upstream of the reaction zone.
 25. A test kit fordetermining an analyte in a sample, comprising a first specified amountof an analyte-binding receptor substance, and a solid phase memberhaving immobilized thereon a second specified amount of saidanalyte-binding receptor substance.
 26. The test kit according to claim25, wherein the ratio between said second amount of analyte-bindingreceptor substance immobilized to the solid phase, and the sum of saidfirst and second amounts of analyte-binding receptor substance is in therange of from about 1:2 to about 1:1000, preferably from about 1:5 toabout 1:100, particularly no more than about 1:20.
 27. The test kitaccording to claim 25 or 26, comprising a lateral flow membrane striphaving said second amount of analyte-binding receptor immobilized in oron a reaction zone of the membrane and having said first amount ofanalyte-binding receptor dissolvably pre-deposited in or on the membraneupstream of the reaction zone.
 28. The test kit according to claim 25 or26, comprising a solid phase well having said second amount of analytebinding receptor immobilized therein and having said first amount ofanalyte-binding receptor dissolvably pre-deposited in the well or inclose contact with the well.